Human Molecular Genetics, 2002, Vol. 11, No. 9 1095-1105
© 2002 Oxford University Press
Expression of Dp260 in muscle tethers the actin cytoskeleton to the dystrophin–glycoprotein complex and partially prevents dystrophy
1Department of Neurology, University of Washington, Seattle, WA 98195, USA and 2Department of Physiology, University of Wisconsin, Madison, WI 53706, USA
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIAL AND METHODSREFERENCES |
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Dystrophin forms a mechanical link between the actin cytoskeleton and the extracellular matrix in muscle that helps maintain sarcolemmal integrity. Two regions of dystrophin have been shown to bind actin: the N-terminal domain and rod domain repeats 11–17. To better understand the roles of these two domains and whether the rod domain actin-binding domain alone can support a mechanically functional link with actin, we constructed transgenic mice expressing Dp260 in skeletal muscle. Dp260, the retinal isoform of dystrophin, lacks the N-terminal domain and a significant portion of the rod domain, but retains the rod domain actin-binding domain. Our results indicate that Dp260 expression restores a stable association between costameric actin and the sarcolemma, assembles the dystrophin–glycoprotein complex, and significantly slows the progression of the dystrophy in the dystrophin-deficient mdx mouse. We assessed the functional integrity of the mechanical link in Dp260 transgenic mdx mice and found that Dp260 muscles showed normal resistance to contraction-induced injury, but dramatic reductions in force generation similar to those found with mdx muscles. Morphologically, Dp260 muscles displayed reduced amounts of inflammation and fibrosis, but still showed a significant, albeit reduced, amount of degeneration/regeneration. These data demonstrate that protection from contraction-induced injury can dramatically ameliorate, but not completely halt, the dystrophic process. We suggest that a non-mechanical defect, attributed to the loss of the N terminus of dystrophin, is likely responsible for the residual dystrophy observed.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIAL AND METHODSREFERENCES |
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Duchenne muscular dystrophy (DMD) is a recessive, X-linked disease caused by defects in the dystrophin gene (1,2). DMD is clinically characterized by progressive muscle degeneration and weakness, which leads to wheelchair dependence by age 11 years and death within the second decade of life from respiratory or cardiac failure (1). Becker muscular dystrophy (BMD) is an allelic disease characterized by a later age of onset and slower rate of disease progression (3). DMD results from mutations that prevent expression of dystrophin, while the milder BMD generally results from mutations that produce partially functional dystrophin (4,5). The mdx mouse also lacks dystrophin and is an animal model for DMD (6). These mice show severe weakness and atrophy in the diaphragm (7,8) and a high susceptibility of all skeletal muscles to contraction-induced injury (9,10).
Dystrophin has been localized in muscle to the cytoplasmic surface of the sarcolemmal membrane in a riblike lattice termed costameres (11,12). Costameres form transverse associations between the sarcolemmal membrane and the contractile elements of the myofiber, and transmit contractile forces through the membrane to the basal lamina (13). The absence of dystrophin leads to changes in the organization of the costameric lattice and an increased susceptibility to contraction-induced damage (14). These observations suggest that dystrophin is important for the organization and stabilization of the sarcolemma and plays a role in protecting muscle fibers from contraction-induced injury (9,15,16). Exactly how dystrophin performs these functions is still being elucidated.
Dystrophin is thought to form a mechanical link between the sarcoplasmic cytoskeleton and the extracellular matrix that helps maintain muscle membrane integrity in part by dissipating the forces of muscle contraction into the extracellular matrix. Dystrophin is a 427 kDa multidomain protein consisting of an N-terminal actin-binding domain (N-ABD), a rodlike domain composed of 24 spectrin-like repeat units and 4 hinge regions, and a C-terminal region that is divided into cysteine-rich and C-terminal domains (Fig. 1A). The N-ABD has been shown to bind actin in vitro (17–20). However, the lower affinity at which the N-ABD binds actin compared with full-length dystrophin (21) and the mild phenotype seen in transgenic mdx mice deleted for the majority of the N-terminal domain (22) implied that there were other actin-binding domains in dystrophin. An additional actin-binding domain was subsequently identified in repeats 11–17 of the central rod domain using in vitro binding assays (21,23,24). While the precise function of the rod domain remains unclear, it has been proposed to participate in force transduction and stabilization of the sarcolemma during muscle contraction. At least some portions of the rod domain are required for dystrophin function, but deletion of up to 20 repeats still produces a functional protein (25). The cysteine-rich and C-terminal domains bind to a multisubunit complex called the dystrophin–glycoprotein complex (DGC) that attaches to laminin in the extracellular matrix (15).
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To determine whether the two actin-binding domains are functionally redundant and if the rod domain actin-binding domain (R-ABD) alone can form a mechanical link to actin that is strong enough to protect the sarcolemma from contraction-induced injury and prevent dystrophy, we constructed transgenic mdx mice expressing Dp260 in skeletal muscle. Dp260 is normally found in the outer plexiform layer (OPL) of the retina and is required for normal retinal electrophysiology (26). The Dp260 transcript is produced from an internal promoter located within intron 29 and has a unique exon 1 spliced in frame to exon 30 (26). This transcript produces a 260 kDa protein that lacks the N-ABD and the first 9
repeats of the rod domain, but contains a unique N terminus of 13 hydrophilic amino acids, repeats 11–24 of the rod domain, and the entire C-terminal region (Fig. 1A). Our data demonstrate that Dp260 is able to restore the link between actin and the extracellular matrix. This linkage protects the Dp260 skeletal muscles from contraction-induced injury, but does not improve force development and only partly prevents muscle degeneration. Therefore, while either actin-binding domain is able to bind actin and protect from contraction-induced injury, the N-ABD appears to play additional roles – perhaps in force transduction or some unknown signaling function – beyond its ability to bind actin. The finding of functional differences between the N-ABD and R-ABD could help explain why patient deletions that encompass only the R-ABD region are scarce and produce only a mild neuromuscular phenotype, while deletions of N-ABD are common with early onset and increased severity (27). |
RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIAL AND METHODSREFERENCES |
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Phenotypic analysis of Dp260 transgenic mdx mice (Dp260/mdx)
To study whether the R-ABD can mechanically link dystrophin to actin and prevent the dystrophic mdx phenotype, transgenic mdx mice were generated that express a Flag-tagged murine Dp260 dystrophin construct (Fig. 1A). Dp260 was used because it contains repeats 11–17 of the rod domain, which has been shown by in vitro binding assays to bind F-actin (21,23,24). In addition, dystrophin constructs with N-terminal truncations are often unstable and poorly expressed (22,27). We speculated that a natural isoform of dystrophin would be more stable and therefore allow for higher transgene expression. F0 mice carrying the Dp260 transgene were crossed onto the mdx background and dystrophin levels were analyzed by western blotting (Fig. 1B). Dp260 expression in the quadriceps and tibialis anterior (TA) was some 4–7-fold higher than that of endogenous dystrophin, but below endogenous levels in the diaphragm (Fig. 1B). Expression was also observed in the extensor digitorum longus (EDL) and soleus muscles (data not shown). The human skeletal
-actin promoter used in this study was not expressed in cardiac muscle (data not shown). Immunofluorescent staining using an antibody against the C-terminus of dystrophin (antibody 18-4) revealed that Dp260 was uniformly expressed and localized correctly to the sarcolemma in both the quadriceps and TA muscles, but displayed low-level, mosaic expression in the diaphragm (Fig. 1C). We have not observed this low-level, mosaic diaphragm expression in other transgenic lines that utilize the human skeletal actin promoter. Therefore, this finding appears unique to the Dp260 transgenic lines and could indicate that this dystrophin construct is especially unstable in the diaphragm. Hematoxylin and eosin-stained limb and diaphragm muscle sections were analyzed in Dp260 transgenic mdx (Dp260/mdx) mice for morphological signs of dystrophy, including necrotic fibers, centrally located nuclei (CN), variation in fiber size, mononuclear cell infiltration and fibrosis (28,29). In the limb muscles of Dp260/mdx mice, few necrotic fibers were observed and little fibrosis and inflammation was evident, although there was some variation in fiber size and an increase in the percentage of CN (Fig. 2A, B). In contrast, the diaphragms of Dp260/mdx mice displayed a dystrophic phenotype, similar to that of age-matched mdx littermates (Fig. 2C). This is most likely due to the low-level, mosaic expression of the transgene in the diaphragm.
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Muscles from dystrophic mdx mice have large numbers of centrally nucleated muscle fibers (28–31). The percentage of CN reflect the degree to which fiber degeneration and regeneration is occurring and is inversely correlated with the ability of a dystrophin molecule to protect muscle from necrosis and regeneration (32–34). Muscle regeneration in mdx mice continues throughout life, but peaks during an acute phase between 5–15 weeks of age (35–37). To estimate the degree of regeneration occurring in Dp260/mdx mice, we determined the percentage of CN in fibers from age-matched C57BL/10, mdx and Dp260/mdx mice on a monthly basis (Fig. 3). C57BL/10 mice displayed less than 2% CN throughout the time course in quadriceps and diaphragm. In the quadriceps, Dp260/mdx mice showed significantly fewer centrally nucleated myofibers (
25%) compared with mdx mice until 20 weeks of age. This result demonstrates that Dp260 is able to partially protect the quadriceps from damage and the resulting regeneration that occurs during the acute phase of the dystrophy. After 20 weeks, the percentage of CN dropped in mdx muscles to a level not different from that of Dp260/mdx mice. This time period corresponds to entry into the chronic phase of the dystrophy, which is characterized by relocalization of some nuclei to the periphery (35). As expected from the poor transgene expression in the diaphragm, there was no significant difference in the percentage of CN in Dp260/mdx and mdx diaphragms.
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The DGC is present in Dp260 transgenic mdx mice
The absence of dystrophin in mdx muscles leads to a dramatic loss of the other members of the DGC (38). We have previously shown that expression of any dystrophin isoform containing the WW and cysteine-rich domains supports stable assembly of the DGC in muscle (8,22,33,34,39). Since Dp260 contains the DGC-assembling portions of dystrophin (34,40,41), we speculated that the DGC would be present in Dp260/mdx mice. To test this hypothesis, microsomes were prepared from C57BL/10 and Dp260/mdx muscles and analyzed by western blotting with antibodies to various DGC members (Fig. 4A). This method allows for the identification of the protein composition of the sarcolemmal membrane from skeletal muscle (42). Dp260/mdx mice contained all analyzed DGC members (Fig. 4A). Microsomes prepared in an identical manner from mdx muscles contain extremely low levels of the DGC components (33). The relative abundance of the various DGC components cannot be compared with the wild-type microsomal preparations because the Dp260 line used in this analysis (line 724) had mosaic expression of the transgene (data not shown). Immunofluorescent staining using antibodies to a subset of DGC members showed that Dp260 correctly localized the DGC to the sarcolemma and confirmed that the DGC components are minimally present in age-matched control mdx muscles (Fig. 4B). The muscle sections used for Figure 4B were from line 723, which expresses Dp260 uniformly. Therefore, the Dp260 protein is able to correctly form and localize the DGC and provide a link to the extracellular matrix.
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Measurement of muscle mechanical properties
Limb and diaphragm muscles of mdx mice display a significant deficit in the ability to generate force and are highly susceptible to contraction-induced injury (8–10,16,43). To quantitatively measure the functional capacity of Dp260 in muscle, mechanical properties were measured in 3- and 6-month-old TA muscles from C57BL/10, Dp260/mdx and mdx mice (Fig. 5). A significant hypertrophy of the TA muscles was observed in both Dp260/mdx and mdx mice (Fig. 5A). Maximum isometric force (P0) development in TA muscles from Dp260/mdx and mdx mice was not significantly different from that of wild-type C57BL/10 muscles (data not shown). However, when P0 values were normalized to the cross-sectional area, the specific force development was significantly lower by about 25% for Dp260/mdx and mdx muscles at both 3 and 6 months of age (Fig. 5B). We also performed a lengthening contraction protocol to test the susceptibility of the TA muscle to contraction-induced injury. This assay revealed a 4–7-fold difference in force deficit between wild-type C57BL/10 and mdx TA muscles (10). Dp260/mdx TA muscles showed no significant difference in force deficit after both lengthening contractions when compared with age-matched C57BL/10 controls (Fig. 5C). These results demonstrated that although Dp260 muscles display normal protection from contraction-induced injury, they were unable to generate normal levels of force.
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Dp260 can form a functional connection to actin in vivo
It has been demonstrated that mechanically peeled wild-type sarcolemma display a well-organized costameric pattern of actin filaments (Fig. 6A), while mdx sarcolemma retain no costameric actin (Fig. 6B and C) (44). The lack of costameric actin associated with mdx sarcolemma is not due to generalized weakness of the sarcolemmal membrane or loss of costameres, since utrophin exhibits a costameric localization in mdx muscles (Fig. 6B). In contrast to the complete absence of costameric actin on mdx sarcolemma (Fig. 6B and C), 10 out of 10 sarcolemma peeled from two different Dp260/mdx mice displayed a well-organized costameric actin pattern (Fig. 6D) and 9 out of 9 also exhibited strong staining with antiserum against repeats 11–14 of the rod domain of dystrophin (rabbit 3; Fig. 6E). The pattern of Dp260 staining was generally costameric, but was more diffusely distributed throughout the sarcolemma than is normally observed with wild-type dystrophin, probably owing to the high expression levels of the transgene. In addition, high Dp260 expression levels likely explain the presence of Dp260 on mechanically peeled myofibers (Fig. 6F). In wild-type muscle, endogenous dystrophin is exclusively found with the peeled sarcolemma (44). These results demonstrate that Dp260 overexpression restores a stable association between costameric actin and the sarcolemmal membrane in vivo. Therefore, the R-ABD can mechanically link the actin cytoskeleton to the sarcolemmal membrane.
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Dp260 has a dominant-negative effect on wild-type dystrophin
F1 Dp260 transgenic wild-type (Dp260,mdx/+) females that express both Dp260 and full-length dystrophin display an increase in the percentage of myofibers with CN compared with age-matched littermates that do not express Dp260 (mdx/+, Fig. 7A). This increase directly correlates with the level of Dp260 transgene expression (Fig. 7B). Thus the quadriceps, which expressed the highest levels of Dp260, display the most centrally nucleated myofibers in wild-type transgenic mice. Despite this increased number of CN, there were no other morphological signs of dystrophy such as necrotic fibers, fibrosis or mononuclear cell infiltration (Fig. 7C). Western analysis showed that expression of Dp260 resulted in lower levels of endogenous dystrophin (Fig. 7B). We have previously observed this effect on endogenous dystrophin levels whenever a transgenic protein is overexpressed relative to wild-type dystrophin levels (45). As with the percentage of myofibers with CN, the decrease in endogenous dystrophin levels correlated directly with the level of Dp260 expression. The TA and quadriceps displayed 80% and 70% decreases in endogenous dystrophin, respectively, while the diaphragm displayed only a 10% decrease. Also, immunofluorescence revealed that fibers highly overexpressing Dp260 displace endogenous dystrophin from the sarcolemma (Fig. 7C). This dominant-negative effect likely results from the ability of the partially functional Dp260 to compete with full-length dystrophin for DGC binding sites when overexpressed. To the best of our knowledge, this is the first observation of a dominant-negative effect due to abnormal dystrophin expression.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIAL AND METHODSREFERENCES |
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The ability of the C-terminal region of dystrophin to bind and assemble the DGC and provide a link to the extracellular matrix has been well characterized. However, the role that the N-terminal and rod domains play in the function of dystrophin and its connection to the cytoskeleton is less well understood. Full-length dystrophin binds to actin with submicromolar affinity and a stoichiometry of 1 dystrophin to 24 actin monomers via multiple lower-affinity actin-binding domains (46). While the two identified actin-binding domains of dystrophin bind actin with similar affinities in vitro (23,47), other biochemical and structural data suggest that the two sites interact with actin filaments through distinct molecular mechanisms. The N-ABD contains a pair of calponin homology domains, which provide the link to F-actin in a number of related proteins, including ß-spectrin,
-actinin, fimbrin and plectin (48,49). Three actin-binding sites have been identified in the N-ABD : ABS1, amino acids 18–27; ABS 2, amino acids 131–148; and ABS3, amino acids 91–117 (reviewed in 17). These sites bind to defined regions of actin (50), suggesting that the N-ABD is a specially designed ‘key’ that fits into a specific ‘lock’ on actin. In contrast, the R-ABD has a broad distribution of positive charges on its surface (basic amino acids) that interact electrostatically with the negative surface charge on actin filaments (23). With this electrostatic design, orientation is not critical and could allow for slippage along the actin filament during muscle contraction.
Studies in mice have shown that only a mild phenotype is associated with deletions of either actin-binding domain (22,32), suggesting that the two actin-binding domains might be functionally redundant. However, our analysis of Dp260, which is deleted for the N-ABD and first 9 repeats of the rod domain, but retains the R-ABD, indicates that they are not functionally equivalent. We have shown that, like the N-ABD, the R-ABD is sufficient to provide a connection to actin in vivo (Fig. 6D, E). The 13 unique amino acids of the N terminus of Dp260 are very basic and likely add strength to the actin-binding capacity of Dp260 through additional electrostatic interactions. While muscles expressing Dp260 are protected from contraction-induced injury, they nonetheless display a subset of the pathological features found in mdx mice. Therefore, specificity of actin binding or some as-yet unidentified role of the N-ABD and/or the first nine rod domain repeats must be critical for dystrophin function.
The importance of the N-ABD and/or repeats 1–9 could explain the milder phenotype observed in transgenic mdx mice expressing the 
N-ABD construct that we reported previously (22). This construct deletes the majority of the N-ABD, but retains the first 44 amino acids of the N terminus and the entire rod domain. The residual N-terminal region, which contains ABS1, could provide weak binding to actin. Moreover, the phenotypic differences between Dp260 and N-ABD raise the possibility that additional rod domain repeats might participate in actin binding, as is the case with the first repeat of ß-spectrin (51) and the first 10 repeats of utrophin (52,53).
The dystrophic pathology observed in mdx mice has been attributed to a mixture of both mechanical and non-mechanical defects caused by the lack of dystrophin and the secondary reduction of the other DGC members (54,55). The mechanical defect results from loss of the link between the cytoskeleton and the extracellular matrix, which renders the sarcolemma susceptible to contraction-induced injury (9,10,16,56). This mechanical defect has been corrected in Dp260/mdx mice, as evidenced by the ability of Dp260 to rescue costameric actin (Fig. 6D, E), assemble the DGC (Fig. 4), and protect the sarcolemma from contraction-induced injury (Fig. 5C). Until now, the non-mechanical defects have been ascribed to the secondary loss of other DGC members such as the sarcoglycans (57) and dystrobrevins (58). However, our data argue for the involvement of an additional non-mechanical defect attributed to the loss of the N terminus of dystrophin.
Our data provide several clues about what might be causing the residual dystrophic phenotype in Dp260/mdx mice. We suggest that the lack of the dystrophin N-terminal regions contributes to the dystrophic process by disrupting the normal cytoskeletal architecture and/or by loss of an as-yet unidentified dystrophin-associated protein-binding site. These disruptions could explain the reduced force development displayed by Dp260/mdx muscles. Dp260 forms a strong bond with the actin cytoskeleton in transgenic muscles (Fig. 6E). However, the different types of actin interactions between N-ABD and R-ABD could presumably alter the overall organization of the cytoskeleton, since the lack of dystrophin in mdx mice has been shown to cause profound cytoskeletal alterations involving many proteins (14). Dp260 is normally expressed only in a non-force-generating tissue, supporting the notion that the N-terminal regions of dystrophin are important for normal force production. Perhaps the ability to transmit forces from the contractile elements via costameric structures to the extracellular matrix requires the specificity of actin binding that the N-ABD provides and/or particular rod domain conformations that are disrupted by deletion of the first 9 repeats. Indeed, in other transgenic mdx mouse studies, we have observed that deletion of 20 of the 24 repeats generates a dystrophin protein that can also prevent contraction-induced injury but does not support normal force development in muscle (25). Furthermore, the N-terminal regions could bind additional proteins needed for force production. The possibility of a novel protein binding to the dystrophin N-terminal domain or the first nine repeats of the rod domain is purely speculative, although precedents exist for protein binding to both the -actinin and ß-spectrin rod domains (59–62). In addition, the homologous actin-binding domains of plectin, fimbrin and -actinin bind to 6ß4 integrin (63), vimentin (64) and CRP1 (65), respectively.
Aside from a role in altering force, altered cytoskeletal architecture could also lead to non-mechanical damage of the muscle fibers. Several non-mechanical mechanisms have been proposed to contribute to the dystrophic process in dystrophin-deficient muscle. These include defects in signaling and/or aberrant calcium homeostasis. Altered calcium homeostasis in dystrophic muscle could result from either mechanical or non-mechanical alterations, or from both. Sarcolemmal tears that result from contraction-induced injuries can clearly lead to localized calcium influx that might initiate a destructive chain of events (66). However, Ca2+ influx through improperly regulated mechanosensitive or non-selective leak channels could occur in the absence of physical damage to the sarcolemma (67–71). Aberrant signaling due to loss of key components of the DGC could potentially lead to such alterations, but we have found no evidence of an altered DGC in Dp260/mdx muscles (Fig. 4). Instead, we suggest that an altered cytoskeletal architecture resulting from loss of the dystrophin N-ABD and/or the first nine spectrin repeats could relax proper regulation of calcium or other ion channels in the absence of significant mechanical damage to the sarcolemma.
Abnormal cytoskeletal architecture could also lead to improper muscle function by disrupting the localization of proteins suspected of responding to mechanical signals, such as the stress-activated protein kinase 3 (72). A variety of other proteins have recently been suggested to be important for the interaction of dystrophin or utrophin with the cytoskeleton through intermediate filaments and microtubules, such as syncoilin, desmuslin and MAST-like proteins (73–75). Aquaporins and sodium channels could also conceivable be altered in Dp260/mdx mice (76). The Dp260/mdx mouse may provide a useful model system to identify the precise mechanisms that contribute to the dystrophic pathology in the absence of mechanical damage to myofibers.
The absence of mechanical damage could also account for the observed reduction in secondary responses such as inflammatory cell infiltration and muscle degeneration. It has been suggested that inflammation can actively contribute to the dystrophic process (77). The lack of fibrosis in Dp260/mdx mice may also be related to the absence of immune cell infiltration, since nude mdx mice that lack most T cells display reduced fibrosis (78). In addition, maintenance of neuronal nitric oxide synthase (nNOS) at the sarcolemma may provide further protection due to the anti-inflammatory actions of nNOS (79). Dp260 appears to slow down the degenerative process similar to what is seen in BMD patients. Reduction of muscle degeneration may prevent or delay exhaustion of the regenerative capacity of satellite cells to repair muscle fibers (80).
A remarkable observation from our studies was that overexpression of Dp260 in wild-type muscles was associated with a dominant-negative effect. This effect likely results from displacement of wild-type dystrophin from the actin cytoskeleton, the DGC, or both, allowing the reduced functional capacity of Dp260 to be manifested. We speculate that overexpression of any truncated dystrophin minigene that displaces endogenous dystrophin, but is not completely functional, could also lead to a dominant-negative effect. No dominant-negative effect was observed in either our N-ABD or Dp71 transgenic mice (22,81). However, neither construct was expressed at greater than wild-type dystrophin levels.
Our data also provide clues to the normal function of Dp260 in the retina. Since Dp260 forms a functional connection to actin in vivo, an interaction with the cytoskeleton is likely important for Dp260 function in the retina. Previous studies have shown that other members of the DGC, such as - and ß-dystroglycan, co-localize with Dp260 in the OPL (82–84). Retinal abnormalities are known to result from mutations that disrupt the link between dystrophin and the DGC, suggesting that dystrophin serves to anchor a form of the DGC to the cytoskeleton in the retina (26,85,86). The retinal defect is thought to result from impaired synaptic transmission between photoreceptors and ON-bipolar cells in the OPL (87). It will be interesting to determine if Dp260 facilitates formation of a DGC-like complex that helps localize signal transduction molecules to the OPL that are important for transmission of light signals.
Our results provide additional evidence for the nature of the mechanical and non-mechanical functions of dystrophin and how defects in either contribute to a dystrophic pathology. We showed that the correction of the mechanical defect, due to the actin-binding ability of the R-ABD, dramatically ameliorates the dystrophic phenotype. A non-mechanical function is suggested for the N-terminal domain of dystrophin that may not be directly related to its ability to bind actin. Elucidation of these additional role(s) will require a better understanding of the nature of this non-mechanical defect in Dp260/mdx mice. Developing a complete understanding of how dystrophin deficiency leads to the dystrophic pathology and the importance of its various domains will help guide development of both genetic and pharmacological therapies for Duchenne muscular dystrophy.
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MATERIAL AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIAL AND METHODSREFERENCES |
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Dp260 transgenic mdx mice
Exons 1–29 from the full-length murine dystrophin cDNA [GenBank accession no. M68859; (88)] were replaced by recombinant PCR with the unique retinal-specific exon 1, isolated from mouse genomic DNA. A Flag epitope (DYKDDDDK) was added in frame to the 5' end of the Dp260 construct by PCR. This Flag-tagged Dp260 cDNA was cloned into an expression vector (previously described in 33) containing the human skeletal
-actin promoter, a splice acceptor from the SV40 VP1 intron, and the SV40 polyadenylation signal. Wild-type C57BL/6x(C57BL/6xSJL) F1 hybrid embryos were injected with the Flag-tagged Dp260 excised expression cassette, and F0 mice were screened by PCR. Three positive F0 lines (723, 724, 596) were backcrossed onto the mdx background. The majority of the studies discussed used line 723 because it showed high-level, uniform expression.
Histological analysis
Skeletal muscles (quadriceps, EDL, TA and diaphragm) were removed from wild-type C57BL/10, mdx, Dp260/mdx, and Dp260,mdx/+ mice at various ages, frozen in liquid-nitrogen-cooled OCT embedding medium (Sakura Finetek USA, Inc.), and cut into 7 µm sections. Sections were stained with hematoxylin and eosin–phloxine and photographed on a Nikon E1000 microscope using the Montage Explorer software (Syncroscopy). The percentage of fibers with CN was determined by dividing the number of muscle fibers with CN by the total number of muscle fibers. 800–1000 fibers were counted per muscle.
Western analysis
Total muscle homogenates were prepared from quadriceps, TA and diaphragm tissues of C57BL/10, mdx and Dp260/mdx mice as previously described (22). Proteins were separated on a 6% SDS–PAGE gel and electrophoretically transferred onto nitrocellulose using a wet transfer apparatus. Membranes were incubated with the monoclonal antibody DYS2 (Novacastra Laboratories, Ltd), washed, and then probed with horseradish peroxidase-conjugated goat anti-mouse antibodies (Jackson ImmunoResearch). Blots were developed using the ECL chemiluminescence system (Amersham). Skeletal muscle microsomes from 10-week-old C57BL/10 and Dp260/mdx mice (line 724) were prepared as described previously (42). KCl-washed microsomes were analyzed by western blot using antibodies previously described (33).
Immunofluorescence
Quadriceps, TA and diaphragm muscles from C57BL/10, mdx, Dp260/mdx, Dp260,mdx/+, and mdx/+ mice were removed, frozen in OCT embedding medium (Sakura Finetek USA, Inc.), and cut into 7 µm sections. Immunofluorescence was performed with the following primary antibodies: 18-4, an affinity-purified polyclonal antibody against the C terminus of dystrophin (81); Rabbit anti-Flag antibody (Sigma); N-term, a polyclonal antibody against the N-terminal domain of dystrophin (34); ß-dystroglycan (Novacastra Laboratories, Ltd.); ,ß,
-sarcoglycan (Novacastra Laboratories, Ltd.); -dystrobrevin 2; or 1-syntrophin (33). After incubation with primary antibodies, cryosections were incubated with either goat anti-rabbit Alexa 488 (for dystrophin, -dystrobrevin 2 and 1-syntrophin antibodies, Molecular Probes) or goat anti-rabbit Alexa 594 (for the Flag antibody, Molecular Probes). ß-Dystroglycan and ,ß,-sarcoglycan are monoclonal antibodies and thus required the use of the Vector MOM Immunodection Kit (Vector Laboratories). Images were collected on a Nikon E1000 microscope under identical conditions using a Spot II CCD camera.
In situ measurements of force and contraction-induced injury
Functional properties of TA muscles from 3- and 6-month-old C57BL/10, mdx and Dp260/mdx mice were measured using methods previously described (10). In brief, TA muscles were maximally stimulated in situ and stretched 1.4 times the optimal fiber length from the plateau of a maximum isometric contraction. The maximum isometric force (P0) was recorded after each lengthening contraction (LC1 and LC2), and damage to the muscle was reported as the force deficit. This value was calculated as the difference between the pre-stretch P0 and the P0 measured after each stretch, and expressed as a percentage of the pre-stretch P0. Specific force was calculated by dividing P0 by the muscle cross-sectional area, determined as previously described (10). At least four TA muscles were measured per strain and time point.
Immunofluorescence analysis of mechanically peeled sarcolemma and myofibers
Inside-out sarcolemma and peeled myofibers were isolated from the EDL muscles of 4-month-old wild-type, mdx and Dp260/mdx mice as previously described (44). Peeled myofibers and sarcolemma were double-stained with Alexa 568–phalloidin (F-actin, Molecular Probes) and either utrophin-specific rabbit 56 antiserum (89) or rabbit polyclonal antiserum to dystrophin amino acids 1–246 (Rabbit 2, N-ABD) or amino acids 1416–1880 (Rabbit 3, rod domain). Rabbit polyclonal antibody staining was detected with Alexa 488 anti-rabbit IgG (Molecular Probes). Confocal microscopy was performed essentially as described (44) in the Keck Center for Biological Imaging at the University of Wisconsin.
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ACKNOWLEDGEMENTS |
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We thank Drs Kevin Campbell and Stan Froehner for generously supplying antibodies. This work was supported by grants from the National Institutes of Health (AR40864 to J.S.C., AR42423 and AR01985 to J.M.E.), and by the Muscular Dystrophy Association USA to J.S.C. and I.N.R. L.E.W. is supported by a National Research Service Award from the National Institutes of Health (AR08630).
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FOOTNOTES |
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* To whom correspondence should be addressed at: Department of Neurology, University of Washington, 1959 NE Pacific Street, Room K243B HSB, Box 357720, Seattle, WA 98195-7720, USA. Tel: +1 206 221 5363; Fax: +1 206 616 8272; Email: JSC5{at}u.washington.edu
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIAL AND METHODSREFERENCES |
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